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Generation of scaffoldless hyaline cartilaginous tissue from human iPSCs.

Yamashita A, Morioka M, Yahara Y, Okada M, Kobayashi T, Kuriyama S, Matsuda S, Tsumaki N - Stem Cell Reports (2015)

Bottom Line: Defects in articular cartilage ultimately result in loss of joint function.The immunodeficiency mice and rats suffered from neither tumors nor ectopic tissue formation.The hiPSC-derived cartilaginous particles constitute a viable cell source for regenerating cartilage defects.

View Article: PubMed Central - PubMed

Affiliation: Cell Induction and Regulation Field, Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto 606-8507, Japan.

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Optimized Protocol for Differentiating hiPSCs toward Chondrocytes(A) Images of hiPSC-derived cells induced in the presence of the indicated supplement on day 14. Top panels: phase view. Bottom panels: GFP fluorescence view. The right panels show images of cells derived from hiPSCs that did not bear the COL11A2-EGFP transgene cultured in the presence of ABTG supplementation. Scale bars, 50 μm.(B) FACS analysis of COL11A2-EGFP-positive cells in the iPSC-derived cell culture in the presence of the indicated supplements on day 14. The error bars denote the means ± SD of three individual experiments. ∗∗p < 0.01.(C) Phase and GFP fluorescence images of the COL11A2-EGFP hiPSC-derived cell culture under ABTG supplementation. Scale bars, 50 μm.(D) Images of the hiPSC-derived particles on day 56 in 3.5-cm dishes. Scale bar, 5 mm.(E) Schematic representation of the protocol for differentiating hiPSCs toward chondrocytes.See also Figure S1.
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fig1: Optimized Protocol for Differentiating hiPSCs toward Chondrocytes(A) Images of hiPSC-derived cells induced in the presence of the indicated supplement on day 14. Top panels: phase view. Bottom panels: GFP fluorescence view. The right panels show images of cells derived from hiPSCs that did not bear the COL11A2-EGFP transgene cultured in the presence of ABTG supplementation. Scale bars, 50 μm.(B) FACS analysis of COL11A2-EGFP-positive cells in the iPSC-derived cell culture in the presence of the indicated supplements on day 14. The error bars denote the means ± SD of three individual experiments. ∗∗p < 0.01.(C) Phase and GFP fluorescence images of the COL11A2-EGFP hiPSC-derived cell culture under ABTG supplementation. Scale bars, 50 μm.(D) Images of the hiPSC-derived particles on day 56 in 3.5-cm dishes. Scale bar, 5 mm.(E) Schematic representation of the protocol for differentiating hiPSCs toward chondrocytes.See also Figure S1.

Mentions: The COL11A2-EGFP hiPSCs were initially differentiated into mesendodermal cells by Wnt3a and Activin A, as previously reported (Oldershaw et al., 2010; Umeda et al., 2012), for 3 days. On day 3, the medium was changed to basal medium supplemented with chondrogenic factors aimed to commit the cells to the chondrocytic lineage. We tested three types of supplementation: A (ascorbic acid), ABT (ascorbic acid, BMP2, and transforming growth factor β1 [TGF-β1]), and ABTG (ascorbic acid, BMP2, TGF-β1, and GDF5). These supplements were added to the basal medium (DMEM with 1% insulin-transferrin-selenium [ITS] and 1% fetal bovine serum [FBS]). Basic fibroblast growth factor (bFGF) was added during the adherent culture (day 3 to day 14) to promote cell proliferation. The hiPSC-derived mesendodermal cells did not form nodules under the conditions of A supplementation, whereas they became focally multilayered and formed nodules under the conditions of ABT or ABTG supplementation on day 14 (Figure 1A). The nodules observed under the conditions of ABTG supplementation specifically exhibited COL11A2-EGFP fluorescence, whereas the nodules formed under the conditions of ABT supplementation did not. Additionally, ABTG produced a significantly higher ratio of COL11A2-EGFP-positive cells than did either A or ATB according to fluorescence-activated cell sorting (FACS) analysis (Figure 1B). The characteristics of human COL11A2-EGFP-positive cells on day 14 may corresponded to those of early precursor cells and chondrocyte-committed cells, as the Col11a2-LacZ (Tsumaki et al., 1996) and Col11a2-EGFP (Hiramatsu et al., 2011) reporter genes were expressed in condensing mesenchymal cells in the limb buds of transgenic mice at 12.5 days postcoitum.


Generation of scaffoldless hyaline cartilaginous tissue from human iPSCs.

Yamashita A, Morioka M, Yahara Y, Okada M, Kobayashi T, Kuriyama S, Matsuda S, Tsumaki N - Stem Cell Reports (2015)

Optimized Protocol for Differentiating hiPSCs toward Chondrocytes(A) Images of hiPSC-derived cells induced in the presence of the indicated supplement on day 14. Top panels: phase view. Bottom panels: GFP fluorescence view. The right panels show images of cells derived from hiPSCs that did not bear the COL11A2-EGFP transgene cultured in the presence of ABTG supplementation. Scale bars, 50 μm.(B) FACS analysis of COL11A2-EGFP-positive cells in the iPSC-derived cell culture in the presence of the indicated supplements on day 14. The error bars denote the means ± SD of three individual experiments. ∗∗p < 0.01.(C) Phase and GFP fluorescence images of the COL11A2-EGFP hiPSC-derived cell culture under ABTG supplementation. Scale bars, 50 μm.(D) Images of the hiPSC-derived particles on day 56 in 3.5-cm dishes. Scale bar, 5 mm.(E) Schematic representation of the protocol for differentiating hiPSCs toward chondrocytes.See also Figure S1.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4375934&req=5

fig1: Optimized Protocol for Differentiating hiPSCs toward Chondrocytes(A) Images of hiPSC-derived cells induced in the presence of the indicated supplement on day 14. Top panels: phase view. Bottom panels: GFP fluorescence view. The right panels show images of cells derived from hiPSCs that did not bear the COL11A2-EGFP transgene cultured in the presence of ABTG supplementation. Scale bars, 50 μm.(B) FACS analysis of COL11A2-EGFP-positive cells in the iPSC-derived cell culture in the presence of the indicated supplements on day 14. The error bars denote the means ± SD of three individual experiments. ∗∗p < 0.01.(C) Phase and GFP fluorescence images of the COL11A2-EGFP hiPSC-derived cell culture under ABTG supplementation. Scale bars, 50 μm.(D) Images of the hiPSC-derived particles on day 56 in 3.5-cm dishes. Scale bar, 5 mm.(E) Schematic representation of the protocol for differentiating hiPSCs toward chondrocytes.See also Figure S1.
Mentions: The COL11A2-EGFP hiPSCs were initially differentiated into mesendodermal cells by Wnt3a and Activin A, as previously reported (Oldershaw et al., 2010; Umeda et al., 2012), for 3 days. On day 3, the medium was changed to basal medium supplemented with chondrogenic factors aimed to commit the cells to the chondrocytic lineage. We tested three types of supplementation: A (ascorbic acid), ABT (ascorbic acid, BMP2, and transforming growth factor β1 [TGF-β1]), and ABTG (ascorbic acid, BMP2, TGF-β1, and GDF5). These supplements were added to the basal medium (DMEM with 1% insulin-transferrin-selenium [ITS] and 1% fetal bovine serum [FBS]). Basic fibroblast growth factor (bFGF) was added during the adherent culture (day 3 to day 14) to promote cell proliferation. The hiPSC-derived mesendodermal cells did not form nodules under the conditions of A supplementation, whereas they became focally multilayered and formed nodules under the conditions of ABT or ABTG supplementation on day 14 (Figure 1A). The nodules observed under the conditions of ABTG supplementation specifically exhibited COL11A2-EGFP fluorescence, whereas the nodules formed under the conditions of ABT supplementation did not. Additionally, ABTG produced a significantly higher ratio of COL11A2-EGFP-positive cells than did either A or ATB according to fluorescence-activated cell sorting (FACS) analysis (Figure 1B). The characteristics of human COL11A2-EGFP-positive cells on day 14 may corresponded to those of early precursor cells and chondrocyte-committed cells, as the Col11a2-LacZ (Tsumaki et al., 1996) and Col11a2-EGFP (Hiramatsu et al., 2011) reporter genes were expressed in condensing mesenchymal cells in the limb buds of transgenic mice at 12.5 days postcoitum.

Bottom Line: Defects in articular cartilage ultimately result in loss of joint function.The immunodeficiency mice and rats suffered from neither tumors nor ectopic tissue formation.The hiPSC-derived cartilaginous particles constitute a viable cell source for regenerating cartilage defects.

View Article: PubMed Central - PubMed

Affiliation: Cell Induction and Regulation Field, Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto 606-8507, Japan.

Show MeSH
Related in: MedlinePlus